Method of producing a recombinant non-glycosylated gp90 of equine infectious anemia virus (EIAV), and product thereof

ABSTRACT

The present invention describes recombinant gp90 envelop protein derived from the equine infectious anemia virus, their corresponding encoding recombinant DNA molecule and the process of production of the recombinant protein produced through genetic engineering techniques, to be used in diagnosis, vaccination or in research.

TECHNICAL FIELD OF THE INVENTION

The present invention refers to the general field of the technology ofthe DNA recombinant proteins, for the production of the gp90 envelopeprotein of Equine Infectious Anemia virus (EIAV), to be used indiagnosis, vaccination, antibody production or in research field.

BACKGROUND TO THE INVENTION

The equine infectious anemia (EIA) is one of the oldest diseases causedby virus, having been described for the first time in France by LIGNEE,Rec. Med. Vet, 20:30, 1843, and recognized as viral disease by VALLEEand CARRE. Acad. Sci., 139:331-333, 1904. The disease affectsexclusively the members of the family Equidae presenting a worldwidedistribution and consequently of great economical importance.

The EIA virus (EIAV) is classified as a lentivirus of the Retroviridaefamily (CHARMAN et al. J. Virol. 19(2):1073-1076, 1976), it is geneticand antigenically related to the other lentiviruses which arecharacterized by causing persistent infection. The EIA has played aspecially important role in comparative virology and in the studies ofthe acquired immunodeficiency syndrome (AIDS). Besides theirmorphological identity, both viruses possess similarities in terms ofnucleotide sequences that code for structural proteins, and they infectthe same cells. These viruses present genetic and antigenic variantsduring persistent infections, which is associated to the immunologicevasion MONTAGNIER et al. Ann. Virol., 135:119-134, 1984, MONTELARO etal. J. Biol. Chem., 259:10539-10544, 1984, RUSHLOW et al. Virology,155:309-321, 1986, STREICHER et al. J. Am. Med. Assoc. 256:2390-23911986, STOLER et al. J. Am. Med. Assoc. 256,2360-2364, 1986 and HAHN

The transmission of EIAV occurs mainly by arthropods vectors(tabanideos) by inoculating the virus into the animal's blood streamwhen feeding themselves (mechanical transmission) justifying the highprevalence of EIA in hot areas favorable to the life cycle of of thesevectors ISSEL et al. Vet. 17:251-286, 1988. EIA can also be transmittedby the placenta and colostro of mare with high virus titers, and byneedles and surgical instruments contaminated with blood COGGINSComparative diagnosis of viral diseases, N.Y., 4:646-658, 1981. Thedisease present the acute forms, subacute, chronic and mainly inaparentor assimptomaticxn lSSEL & COGGINS, J. Am. Vet. Med. Assoc.174(7):727-33, 1979, and the most prominent signs are the feverishepisodes, anemia hemolitica, anorexia, fast weight loss and ventraledema.

Considering the high prevalence of assymptomatic carriers, the nonconclusive clinical diagnosis and the possibility to confuse with otherdiseases as the trypanosomiases, piroplasmose, leptospirose, hepatitisand endoparasitoses the laboratory diagnosis plays a decisive role inthe control and prevention of EIA.

The accepted way to diagnose the presence of EIA has been to detect thepresence of antibodies specific for the disease in the serum of affectedanimals using the Coggins or agar gel diffusion test described in U.S.Pat. No. 3,929,982 and U.S. Pat. No. 3,932,601. In the Coggins test, aprepared antigen is placed alongside the senum to be tested in an agaror gel medium. If EIA antibodies are present in the test serum, theywill diffuse toward the antigen forming a precipitin line in the agarmedium where they eventually meet. The antigen is prepared, using spleenof infected horses COGGINS & NORCROSS Cornell. Vet. 60(2):330-5, 1970 orin culture of horse leucocytes NAKAJIMA & USHIMI Infect Immun,3(3):373-7, 1971.

This methodology is inherently insensitive in that the EIA antigen maybe contaminated with non-EIA antigens during its preparation. Antibodiesagainst non-EIA antigens may be present in the test serum and can reactEven if the prepared EIA viral antigen can be purified, the Coggins testis labor intensive and demanding of considerable expertise ininterpretation of results. The Coggins test procedure is also slow toyield results, it takes twenty-four to forty-eight hours for theformation of clearly visible precipiting lines.

Porter, U.S. Pat. No. 4,806,467, discloses a method for detecting theEIA virus using a complete enzyme-linked immunoabsorbent assayincorporating a purified viral antigen and a monoclonal antibody. Toobtain the antigen, the EIA virus must first be cultured. The antigen isthe p26 core protein of the EIA virus and is obtained through(purification of the cultured virus by a variety of means) well known inthe art. The technique of culturing a virus increases the likelihoodthat the assay will yeild false positive results since the virus may becontaminated with other forms of protein. Addtionally, the EIA virus ishard to culture, making the Porter approach difficult for large scaleproduction.

The use of a synthetic peptide in an enzyme linked (immunosorbent assay)for the detection of human immunodeficiency virus (HIV) is disclosed inShoeman, R. L. et al, Analytical Biochemistry 161:370-379 (1987). HIVand the EIA virus are members of the retrovirus family but havedissimilar structures and distinct amino acid sequences.

The main component of these preparations is therefore a protein of thevirai capsid whose molecular weight is 26 KDa, (denominated p26) This isthe most abundant protein of the viral particle PAREKH et al. Virology,107:520-525, 1980, GELDERBLON, AIDS 5:617-638, 1991, and it is highlyconserved within the variant samples of the isolated viruses HUSSAIN etal. J. Virol. 61:2956-2961, 1987, SALINOVICH et al. J. Virol. 57:71-80,1986. and infected horses present specific antibodies anti-p26.

Darrel & Peisheng, the U.S. Pat. No. 5.427,907, discloses a method touse a synthetic peptide as the antigen in an immunoassay for thedetection of antibodies against the equine infectious anemia virus inthe serum of horses. This procedure include only the search of someepitopes of virus proteins.

It is an object of the present invention to describe the recombinantgp90 envelope protein from AIEV, their corresponding encodingrecombinant DNA molecule and the process of production of therecombinant gp90 envelope protein produced through techniques of geneticengineering, to be used for diagnosis, vaccination or in research.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many attendant advantages of theinvention will be better understood upon a reading of the followngdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 shows the vector used for the expression of the recombinant gp90envelope protein

FIG. 2 shows the amino acid sequence of the recombinant gp90 envelopeprotein

FIG. 3 shows the pattern of hydrophilicity of the recombinantgp90envelope protein

FIG. 4 shows a print of SDS-PAGE of expressed and purified recombinantgp90 protein in E. coli

DETAILED DESCRIPTION OF THE INVENTION

The methodology used for the production of the recombinant gp90envelopeprotein consists of the cloning and expression, in microorganisms, ofthe DNA corresponding to the gene that codes the protein gp90 of the EIAusing the methodology of the genetic engineering.

In order to better understand this invention the following examples, forillustrative purposes only, are described. The examples illustrate thepresent invention and are not intended to limit it in spirit or scope.The process can be understood better through the following descriptionin consonance with the examples.

EXAMPLE 1 Virus Multiplication (1)

For multiplication of the virus cells from equine derme were used (ED)or other cells that allow replication of the virus AIE.

EXAMPLE 2 Extraction of Genomic DNA (2)

To obtain the genomic DNA (pro virus) the of cells were washed withsaline STE (Tris-HCl 10-10.5 mM pH 8-8.5 NaCl 0.1 M, EDTA 1-1.5 mM). Thefollowing stage was the lysis of the cells with a solution of STEduodecil sodio (SDS sulfato) and proteinase K,(Sigma, USES) for 10-30minutes, incubated for 50° C.-55° C. for 12-18 hours. The precipitationof the DNA was made with isopropanol and centrifuged. The pellet washomogenized in water transferred to eppendorf tube and incubated for 60°C.-65° C. for 1-2 hours for complete breakup of the DNA

EXAMPLE 3 DNA Amplification (3)

The amplification of the DNA (3) starting from the proviral DNA obtainedin the stages 1, 2, or starting from the vector that contains the clonedDNA of the gene GP-90 it wascarried out using as primers the followingspecific oligonucleotides (SEQ ID NO: 1) 5′CAGTGGATCCT TCCCGGGGTGTAGA-3and 5′CAATCTGCAGAATTAGTCCAGTGTTAG-3′. Those oligonucleotides were drawnto amplify, through polymerase chain reaction (PCR), the DNA region thatencodes the corresponding fragment of the gp90 envelope protein. Theprimers also contains the sites for the restriction enzymes BamH-1 andHind III. TCCCGGGGTGTAGA-3 and 5′CAATCTGCAGMTTAGTCCAGTGTTAG-3′. Thoseoligonucleotides were drawn to amplify, through polymerase chainreaction (PCR), the DNA region that encodes the corresponding fragmentof the gp90 envelope protein. The primers also contains the sites forthe restiction enzymes BamH-1 and Hind III.

The PCR reaction was performed with Taq polymerase buffer (50 mM KCl,100 mM Tris-HCl pH 9.0-9.5, 1.5-2.5 mM MgCl₂ and 1-2% triton X-100),0.1-1 U of Taq polymerase (Promega, E.U.A., Cat. no. M186A), 0.5-1.5 mMMgCl₂, 20-50 mM of each nucleotide (dATP,dCTP,dGTP,d dTTP) 10-30 μmolesof each primer, and 0.01a 0.1 μg cDNA and H₂O q.s.p. 50-100 □. Thereaction was performedin 1-2 cycles at 94-96° C./1-2 mi 53 to 55° C./1-2min.; 70-72° C./1-2 min; 30 cycles at 94-96° C./1 to 2 min; 36-38°C./1-2 min; 70-72° C./1-2 min and more 1 cycle to 94-96° C./1-2 min;36-38° C./1 to 2 min; 70-72° C./10-15 min.

The PCR product was fractionated by electrophoresis in 1.5-2.0% agarosegel and purification was made by cutting out the band of the gel. Theband was diluted in 2-3 volumes of Nal solution (Nat 8M+0.022 M DTT) andsodium phosphate buffer (1M pH 6.0-6.5) and incubated for 5-10 min. at50-56° C. Glass beads were added to the suspension, mixed incubated 1-5min. at room temperature and centrifuged 10-30 seconds The spheres werewashed with ethanol buffer (75% of ethanol, 0.01 M Tris-HCl, pH 7.0-7.6,0.01 M EDTA, pH 8.0-8.5). The DNA was eluted from the glass spheres withbuffer (Tris pH 7.0-7.4 10 mM, 1-3 mM EDTA) at 50-56° C. for 1-5 min.

EXAMPLE 4 Cloning (4)

The PCR product was digested with enzyme Hind III with 10-20 U of HindIII (Biolabs, England) plus 3-5 μl buffer (Promega,EUA) in 30-50 μlvolume of H₂O. The reactions were incubated at 37° C. for 2-4 h. Afterthis time 10-20 U of Bam Hi (Biolabs, England) plus 5-10 μl of react IIIbuffer (BRL, USA) were added to a final 50-100μl volume of H₂O dd and itwas incubated at 37° C. for 2-4.h. For cloning of the PCR product intoplasmid PDS-56 (FIG. 1), the vector vas digested with 10-20 U of enzymeHind III (Promega, USA), 2-5 μl buffer I B (Promega, E.U.A.) in 20-50 μlfinal volume of H₂O, and incubation at 37° C. for 2-4h. To the reactionwas added 10-20 U of the enzyme Bam HI (Promege, USA), 5‥10 μl of reactIII (BRL, E.U.A.), in 50-100 μl final volume of H₂O, and incubation at37° C. for 2-4 h. The product of this digestion was resolved in a 1%TAE-agarose gel electrophoresis. The band corresponding to the digestedplasmid was cutted out of the gel, tranferred to a Eppendorf tube (1.5ml) and pufigied.

In the ligation reaction 20-50 μg of the DNA fragment insert was addedto 5-15 μg of the vector DNA, plus 0.5-2.0 U of T4 Ligase (Promega,USA), 5 mM ATP (Promega, E.U.A.), ligation buffer (Promega, E.U.A.), H₂Odd qsp 15 μl, with incubation at 14-16° C. (BOD, FANEN, Brazil) for12-18 h.

EXAMPLE 5 Transformation (5)

The bacterial transformation was done with Escherichia coli by addingthe ligation reaction completed to 40-60 μl volume buffer (Tris 10 mM pH7.2-7.4. EDTA 1 mM) to 100 μl of competent bacteria suspension. Thetubes were slightly rotated and immediately incubated on ice bath for20-40 min . After that, they were submitted to a thermal shock at 40-42°C. for 1-3 min. and kept on ice bath for further 20-40 seconds. LBmedium (Bacto triptona 1% p/v, extract of yeast 0.5% p/v, NaCl 171 mM)without antibiotic was added at double volume and incubated at 37° C.for 1-2h. The bacteria were pelleted, homogenized in LB and inoculatedin Petri dish plates with LB agar (agar 1.5% p/v, yeast extract 0.5%p/v, triptone 0.1% p/v, NaCl 0.5% piv pH 7.2-7.5) with 50-200 μg/mlampicillin and 20-100 μg/ml kanamycin. The plates were incubated at 37°C. for 15-24 h. For the selection of the positive clones they were grownin LB with 50-200 μg/ml ampicillin and 20-100 □g/ml kanamycin at 37° C.under agitation for 15-20 h. After incubation a PCR using specificprimers of the vector (for amplification of the area corresponding toinsert) being the primer (sense) 5′-TTCATTAAAGAGGAGAAATT-3′ (SEQ ID NO:3) and primer (anti-sense)5′-CTATCAACAGGAGTCCAAGC-3′ (SEQ ID NO: 4). Thereaction was made with Taq. polymerase buffer 10X (KCl 500 mM,:Tris-HCl100 mM pH 9.0-9.5, MgCl₂ 15-25 mM and triton X-100 1-2%), 0.5-1.0 U ofTaq polymerase (Promega, USA), 0.5-1.5 mM MgCl₂, 20-50mM of eachnucleotide (dATP, dCTP, dGTP, DTTP), 10-30pmoles of each primer, 0.5-1μl of bacteriai suspension and H₂Odd sterile qsp 20-40 μl. The reactionwas processed with 1-3 cycles of 94-96° C./5 min., 50-55° C./1-2 min.,70-72° C./1-2 min.; 30 cycles of 94-96° C./30-45 seg., 45-50° C./30-45seg., 70-72° C./30-45 seg. and 1 cycle of 94-96° C./1-2 min., 45-50°C./1-2 min., 70-72° C./10-15 min. The product of this reaction wasfractionated through 1-2%.agarose gel electrophoresis.

EXAMPLE 6 Sequencing (6)

The positive clones were sequenced to confirm the sequence of FIG. 2 andpresents the hydrofobicity profile as showed in FIG. 3.

EXAMPLE 7 Protein Production (7)

The positive clones were used for production of protein and they weregrown in LB medium with 50-200 μg/ml ampicillin, 50-200 of Kanamycinμg/ml and incubated at 37° C. under agitation until the optical density(OD 600 nm) of 0.5-4.7. Then, for the induction of the protein,IPTG(Isopropyl-β-D-thiogalactpyranoside) to 0.2-0.4 M was added andincubated for 3-5 h. The bacteria vwere centrifuged, the supernatant wasdiscarded and the pellet homogenized in buffer A (guanidine-HCl 5-6 M,sodium phosphate 0.1-0.2 M, Tris 0.01-0.02 M pH 7.8-8.0) with agitationfor 1-2 h. A polyacrylamide gel shows the expression in the bacteria.

EXAMPLE 8 Protein Purification (8)

After the centrifugation the supernatant was applied to a-column withNi-NTA (nickel chelate) resin. For purification of the protein thecolumn was washed sequentially with buffer A, buffer B (Urea 78 M,phosphate of sodium 0.1-0.2 Tris 0.01-0.02 M pH 7.8-8.0) and with bufferC (Urea 7-8 M, phosphate of sodium 0.1-0.2 M, Tris 0.01-0.02 M pH7.0-7.2). The protein was eluter with buffer D (Urea 7-8 M, sodiumphosphate 0.1-0.2 M, Tris 0.01-0.02 M pH 5.0-5.2) and sequentially withUrea 7-8 M, phosphate of sodium 0.1-0.2 M, Tris 0.01-0.02 M pH 40-4.2.Fractions were collected and 50 μl of each fraction was diluted vN insample buffer, heated for 10 min. and submitted to electrophoresis inpolyacrylamida gel (SDS-PAGE). The gel was analyzed for the presence ofthe fraction that just contained the band corresponding to the purifiedrecombinant protein. (FIG. 4)

While the present invention has been described in connection withexamples, it will be understood that modifications and variationsapparent to those ordinary skill in the art are within the scope of thepresent invention.

5 1 25 DNA Artificial Sequence Description of Artificial Sequenceprimer1 cagtggatcc ttcccggggt gtaga 25 2 27 DNA Artificial SequenceDescription of Artificial Sequenceprimer 2 caatctgcag aattagtcca gtgttag27 3 20 DNA Artificial Sequence Description of Artificial Sequenceprimer3 ttcattaaag aggagaaatt 20 4 20 DNA Artificial Sequence Description ofArtificial Sequenceprimer 4 ctatcaacag gagtccaagc 20 5 321 PRT equineinfectious anemia virus 5 His His His His His His Ser Phe Pro Gly CysArg Pro Phe Gln Asn 1 5 10 15 Tyr Phe Ser Tyr Glu Thr Asn Arg Ser MetHis Met Asp Asn Asn Thr 20 25 30 Ala Thr Leu Leu Glu Ala Tyr His Arg GluIle Thr Phe Ile Tyr Lys 35 40 45 Ser Ser Cys Thr Asp Ser Asp His Cys GlnGlu Tyr Gln Cys Lys Lys 50 55 60 Val Asn Leu Asn Ser Ser Asp Ser Ser AsnSer Val Arg Val Glu Asp 65 70 75 80 Val Thr Asn Thr Ala Glu Tyr Trp GlyPhe Lys Trp Leu Glu Cys Asn 85 90 95 Gln Thr Glu Asn Phe Lys Thr Ile LeuVal Pro Glu Asn Glu Met Val 100 105 110 Asn Ile Asn Asp Thr Asp Thr TrpIle Pro Lys Gly Cys Asn Glu Thr 115 120 125 Trp Ala Arg Val Lys Arg CysPro Ile Asp Ile Leu Tyr Gly Ile His 130 135 140 Pro Ile Arg Leu Cys ValGln Pro Pro Phe Phe Leu Val Gln Glu Lys 145 150 155 160 Gly Ile Ala AspThr Ser Arg Ile Gly Asn Cys Gly Pro Thr Ile Phe 165 170 175 Leu Gly ValLeu Glu Asp Asn Lys Gly Val Val Arg Gly Asp Tyr Thr 180 185 190 Ala CysAsn Val Arg Arg Leu Asn Ile Asn Arg Lys Asp Tyr Thr Gly 195 200 205 IleTyr Gln Val Pro Ile Phe Tyr Thr Cys Thr Phe Thr Asn Ile Thr 210 215 220Ser Cys Asn Asn Glu Pro Ile Ile Ser Val Ile Met Tyr Glu Thr Asn 225 230235 240 Gln Val Gln Tyr Leu Leu Cys Asn Asn Asn Asn Ser Asn Asn Tyr Asn245 250 255 Cys Val Val Gln Ser Phe Gly Val Ile Gly Gln Ala His Leu GluLeu 260 265 270 Pro Arg Pro Asn Lys Arg Ile Arg Asn Gln Ser Phe Asn GlnTyr Asn 275 280 285 Cys Ser Ile Asn Asn Lys Thr Glu Leu Glu Thr Trp LysLeu Val Lys 290 295 300 Thr Ser Gly Val Thr Pro Leu Pro Ile Ser Ser GluAla Asn Thr Gly 305 310 315 320 Leu

What is claimed is:
 1. A process for producing the recombinant gp 90Equine Infectious Anemia envelope protein consisting of an amino acidsequence of SEQ ID NO: 5 comprising culturing in E. coli cell underconditions whereby said protein is produced.
 2. The process according toclaim 1 wherein said amino acid sequence is not glycosylated.
 3. Anamino acid sequence consisting of SEQ ID NO:5 produced by a process ofclaim
 1. 4. An amino acid sequence consisting of SEQ ID NO:5 produced bya process of claim
 2. 5. An amino acid sequence consisting of SEO IDNO:5 which is not glycosylated.